Pyrrolopyrimidine derivatives, their production

ABSTRACT

A process of making a compound of the formula ##STR1## wherein the ring A is a pyrrole or pyrroline ring, X is an amino group or a hydroxyl group, Y is a hydrogen atom, an amino group or a hydroxyl group, R is a hydrogen atom, a fluorine atom, an alkyl group, an alkenyl group or an alkynyl group, --COOR 1  and --COOR 2  are independently carboxyl groups which may be esterified and n is an integer of 2 to 4, and R may be different in each of the n repeating units, and salts thereof have excellent antitumor effects, and can be used as antitumor agents in mammals.

This application is a division of Ser. No. 07/824,106 filed Jan. 22,1992, now U.S. Pat. No. 5,296,600 which is a division of Ser. No.07/578,258 filed Sep. 6, 1990, now U.S. Pat. No. 5,106,974; which is adivision of Ser. No. 07/326,901 filed Mar. 21, 1989 now U.S. Pat. No.4,997,838.

This invention relates to the novel pyrrolopyrimidine derivatives whichare useful as anti-tumor agents, the production and utilization thereof.

Folic acid is a carrier of a C1 unit in a living body, derived fromformic acid or formaldehyde, acting as a coenzyme in various enzymaticreactions such as those in biosynthesis of nucleic acid, in metabolismof amino acids and peptides and in generation of methane. Particularlyin biosynthesis of nucteic acid, folic acid is essential for formylationin the two pathways, i.e. the purine synthetic pathway and the thymidinesynthetic pathway. Usually folic acid is required to be transformed intoits activated coenzyme form by reduction in two steps before it becomesbiologically active. Amethopterin (methotrexate: MTX) and the relatedcompounds are known to inhibit the reduction from dihydrofolic acid intotetrahydrofolic acid by coupling strongly with the dominant enzyme inthe second step (dihydrofolic acid reductase). These drugs have beendeveloped as antitumor drugs because they may disturb the DNA synthesisand consequently cause cell death, and are currently regarded of majorclinical important. On the other hand, a novel tetrahydroaminopterinantitumor agent (5,10-dideaza-5,6,7,8-tetrahydroaminopterin: DDATHF) hasbeen reported which, unlike the drugs described above, does not inhibitdihydrofolic acid reductase and the main mechanism of which consists ininhibition of glycinamide ribonucleotide transformylase required in theinitial stage of purine biosynthesis [Journal of Medicinal Chemistry,28, 914 (1985)].

Various studies are now being conducted on therapy for cancer, and whatis expected strongly is the development of drugs which are moreeffective and have toxicities highly specific to cancer cells based onsome new mechanism. The antitumor agent MTX, the action mechanism ofwhich consists in antagonism against folic acid, is clinically usedwidely, though the therapeutic effect is still unsatisfactory because ithas relatively strong toxicity with little effect on solid cancer.

As the result of the inventors' researches under the circumstancesdescribed above, they have found out that novel pyrrolopyrimidinederivatives have toxicities highly specific to tumor cells and excellentantitumor effects, and completed this invention.

This invention relates to

(1) A compound of the formula (I) ##STR2## wherein the ring A is apyrrole or pyrroline ring, X is an amino group or a hydroxyl group, Y isa hydrogen atom, an amino group or a hydroxyl group, R is a hydrogenatom, a fluorine atom, an alkyl group, an alkenyl group or an alkynylgroup, --COOR¹ and --COOR² are independently carboxyl groups which maybe esterified and n is an integer of 2 to 4, and R may be different ineach of the n repeating units, and salts thereof,

(2) A method for production of the compounds (I) or salts thereofcharacterized in that a compound of the formula (II) ##STR3## whereinthe ring A is a pyrrole or pyrroline ring, X is an amino group or ahydroxyl group, Y is a hydrogen atom, an amino group or a hydroxylgroup, R is a hydrogen atom, a fluorine atom, an alkyl group, an alkenylgroup or an alkynyl group, and n is an integer of 2 to 4, and R may bedifferent in each of the n repeating units, a reactive derivative at thecarboxyl group, or a salt thereof, and a compound of the formula (III)##STR4## wherein --COOR¹ and --COOR² are independently carboxyl groupswhich may be esterified, or a salt thereof, are allowed to react.

(3) A compound of the formula (IV) ##STR5## wherein the ring A is apyrrole or pyrroiine ring, X is an amino group or a hydroxyl group, Y isa hydrogen atom, an amino group or a hydroxyl group, R is a hydrogenatom, a fluorine atom, an alkyl group, an alkenyl group or an alkynylgroup, --COOR³ is a carboxyl group which may be esterified and n is aninteger of 2 to 4, and R may be different in each of the n repeatingunits, and salts thereof.

(4) Anti-tumor agents containing the compounds (I) or salts thereof.

When X or Y in the formulas described above is a hydroxyl group, each ofthe compounds (I), (II) and (IV) may exist as an equilibrium mixture ofthe respective tautomers. The following partial structural formulas showthe sites of the structure which are subject to tautomerism, and theequilibrium between the tautomers is illustrated in the following.##STR6##

For the convenience of description, only the hydroxyl forms and thecorresponding names are described throughout this specification, but thecorresponding oxo forms are always included.

There may be two or more asymmetric centers in the compounds (I) of thisintention, and the absolute configuration at all of the asyrmmetriccarbon atoms may be the S, R or S-R mixed form, except that the absoluteconfiguration at the asymmetric carbon atom in the side chain derivedfrom glutamic acid is always S(L). Therefore the compounds (I) may havetwo or more diastereomers which, if necessary, can easily be separatedfrom each other by a routine method for separation and purification. Allof the diastereomers which can be separated by such a method areincluded in this invention.

Alkyl groups represented by R in the formulas described above includealkenyl groups having 1 to 3 carbon atom(s) each (e.g. methyl, ethyl,propyl, isopropyl groups). Alkenyl groups represented by R in theformulas described above include alkenyl groups having 2 to 3 carbonatom(s) each (e.g. vinyl, 1-methylvinyl, 1-propenyl, allyl, allenylgroups). Alkynyl groups represented by R in the formulas described aboveinclude alkynyl groups having 2 to 3 carbon atom(s) each (e.g. ethynyl,1-propynyl, propagyl groups). Carboxyl groups in the carboxyl groupswhich may be esterified, represented by --COOR¹, --COOR² and --COOR³include carboxyl groups which may be esterified by alkyl groups having 1to 5 carbon atom(s) each, benzyl groups which may be substituted orphenyl groups which may be substituted. The alkyl groups include methyl,ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl,n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl and tert-pentyl. The benzylgroups which may be substituted include benzyl, nitrobenzyl,methoxybenzyl groups and so on. The phenyl groups which may besubstituted include phenyl, nitrophenyl, methoxyphenyl groups and so on.

In the following the method for production of the compounds (I) of thisinvention is explained.

The compounds (I) can be obtained by acylation of glutamic acidderivatives shown by the formula (III) with carboxylic acids shown bythe formula (II) or reactive derivatives thereof. The acylation may beperformed, for example, by acylation of the compound (III) with thecompound (II) in the presence of carbodiimide, dephenylphosphoryl azideor diethyl phosphoro cyanidate. Generally about 1 to 20 mole equivalent,preferably 1 to 5 mole equivalent of the compound (III) relative to thecompound (II) is used. Generally about 1 to 25 mole equivalent,preferably about 1 to 5 mole equivalent of a carbodiimide relative tothe compound (II) is used. As the carbodiimide, dicyclohexylcarbodiimideis preferable for practical use, but other carbodiimides such asdiphenylcarbodiimide, di-o-tolylcarbodiimide, di-p-tolylcarbodiimide,di-tert-butylcarbodiimide,1-cyclohexyl-3-(2-morpholinoehtyl)carbodiimide,1-cyclohexyl-3-(4-diethylaminocyclohexyl)carbodiimide,1-ethyl-3-(2-diethylaminopropyl)carbodiimide and1-ethyl-3-(3-diethylaminopropyl)carbodiimide may be used. The acylationis preferably performed in the presence of a suitable solvent, and suchsolvents include water, alcohols (e.g. methanol, ethanol, etc.), ethers(e.g. dimethyl ether, diethyl ether, tetrahydrofuran, dioxane,monogtyme, diglyme, etc.), nitriles (e.g. acetonitrile, etc.), esters(e.g. ethyl acetate, etc.), halogenated hydrocarbons (e.g.dichloromethane, chloroform, carbon tetrachloride, etc.), aromatichydrocarbons (e.g. benzene, toluene, xylene, etc.), acetone,nitromethane, pyridine, dimethylsulfoxide, dimethylformamide,hexamethylphospholamide, sulfolane, and the suitable mixtures of two ormore of these solvents. The reaction is allowed to proceed generally ata pH ranging from 2 to 14, preferably at a pH ranging from about 6 to 9,at a temperature ranging from about -10° C. to the boiling point of thesolvent used (up to about 100° C.), preferably at a temperature rangingfrom about 0° to 50° C., for about 1 to 100 hours. The pH of thereaction mixture is adjusted, if necessary, by addition of an acid (e.g.hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, aceticacid, etc.), a base (e.g. sodium alcoholate such as sodium methylate andsodium ethylate, hydroxides of alkali metals or of alkali earth metalssuch as sodium hydroxide, potassium hydroxide, lithium hydroxide, bariumhydroxide, carbonates or bicarbonates of alkali metals or of alkaliearth metals such as sodium carbonate, potassium carbonate, bariumcarbonate, calcium carbonate and sodium biccarbonate, amines such astrimethylamine, triethylamine, triethanolamine and pyridine), or abuffer (e.g. phosphate buffer, borate buffer, acetate buffer, etc.). Thereaction can proceed more advantageously in the presence of a catalystwhich promotes acylation. Such catylysts include base catalysts and acidcatalysts. The base catalysts include tertiary amines (e.g. aliphatictertiary amines such as triethylamine; aromatic tertiary amines such aspyridine, α-, β- or γ-picoline, 2,6-lutidine, 4-dimethylaminopyridine,4-(1-pyrrolidinyl)pyridine, dimethylaniline and diethylaniline), andsuch acid catalysts include Lewis acids [e.g. anhydrous zinc chloride,anhydrous aluminum chloride (AlCl₃), anhydrous ferric chloride, titaniumtetrachloride (TiCl₄), tin tetrachloride (SnCl₄), antimonypentachloride, cobalt chloride, cupric chloride, boron trifluoride ethylether complex, etc.]. Among the catalysts described above,4-dimethylaminopyridine or 4-(1-pyrrolidinyl)pyridine is preferable inmany cases. The suitable amount of the catalyst is such that is enoughto oromote the acylation being generally about 0. 01 to 10 moleequivalent, preferably about 0.1 to 1 mole equivalent relative to thecompound (II). The reactive derivatives of carboxylic acids obtained bythe reaction at the carboxyl group, used for the acylation include acidhalides (e.g. fluoride, chloride, bromide, iodide), acid anhydrides(e.g. iodoacetic acid anhydride, isobutyric acid anhydride), mixed acidanhydrides with monoalkylcarbonic acid esters (e.g. mono-methylcarbonicacid ester, monoethylcarbonic acid ester, monopropylcarbonic ester,mono-iso-propylcarbonic acid ester, monobutylcarbonic acid ester,mono-iso-butylcarbonic acid ester, mono-sec-butylcarbonic acid ester,mono-tert-butylcarbonic acid ester), active esters (e.g. cyanomethylester, carboethoxymethyl ester, methoxymethyl ester, phenyl ester,o-nitrophenyl ester, p-nitrophenyl ester, p-carbomethoxyphenyl ester,p-cyanophenyl ester, thiophenyl ester), acid azidest mixed acidanhydrides with phosphoric acid diesters (e.g. dimethyl phosphate,diethyl phosphate, dibenzylphosphate, diphenylphosphate), and mixed acidanhydrides with phosphorous acid diesters (e.g. dimethyl phosphite,diethyl phosphite, dibenzyl phosphite, diphenyl phosphite), of thecarboxylic acid (II). For acylation with such a reactive derivative, thesolvent, the catalyst and the reaction temperature are the same as foracylation in the presence of the carbodiimide described above.

For production of the compound (I-1) in which --COOR¹ and --COOR² in theformula of the compound (I) are carboxyl groups, it is desirable thatthe compound in which --COOR¹ and --COOR² in the formula of the compound(III) are esterified carboxyl groups is allowed to react with thecompound (II) followed by deesterification by per se known degradationor catalytic reduction. Such degradation can be performed by hydrolysisunder basic conditions (method A), hydrolysis under acidic conditions(method B-1) or hydrolysis under acidic nonaqueous conditions (methodB-2). Bases used in the method A include metal alkoxides such as sodiummethoxide, sodium ethoxide, sodium butoxide and potassium butoxide,metal hydroxides such as sodium hydroxide, potassium hydroxide, lithiumhydroxide and barium hydroxide, and amines such as ammonia,triethylamine and pyridine. Acids used in the method B-1 include mineralacids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid and phosphoric acid, and organic acids such as trifluoroaceticacid, trichloroacetic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and camphorsulfonic acid. Catalysts used in themethod B-2 include mineral acids such as hydrogen chloride, hydrogenbromide, perchloric acid, sulfuric acid, nitric acid and phosphoricacid, organic acids such as trifluoroacetic acid, trichloroacetic acid,methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid andcamphorsulfonic acid, and Lewis acids such as anhydrous zinc chloride,anhydrous aluminum chloride (AlCl₃), anhydrous ferric chloride, titaniumtetrachloride (TiCl₄), tin tetrachloride (SnCl₄), antimonypentachloride, cobalt chloride, cuprio chloride and boron trifuluorideethyl ether complex. Degradation is performed in a suitable solvent at atemperature ranging from 0° C. to the boiling point of the solvent,preferably at 10° to 80° C. for 30 minutes to 2 days. The solvent usedfor the reaction by the method A or by the method B-1 may be water,methanol, ethanol, propanol, butanol, ethyleneglycol, methoxyethanol,ethoxyethanol, tetrahydrofuran, dioxane, monoglyme, diglyme, pyridine,dimethylformamide, dimethylsulfoxide or sulfolane, or a suitable mixtureof two or more of these solvents; the solvent used for the reaction bythe method B-2 may be ethyl acetate, dimetyl ether, diethyl ether,tetrahydrofuan, dioxane, monogtyme, diglyme, dichloromethane,chloroform, carbon tetrachloride, acetonitrile, benzene, toluene,xylene, nitromethane or pyridine, or a suitable mixture of two or moreof these solvents. The catalytic reduction (method C) is performed in asuitable solvent at a temperature ranging from about -40° C. to theboiling point of the solvent used, preferably at about 0° to 50° C. Thesolvents used include water., alcohols (e.g. methanol, ethanol,propanol, iso-propanol, butylalcohol, sec-butylalcohol,tert-butylalcohol, ethyleneglycol, methoxyethanol, ethoxyethanol),acetic acid esters (e.g. methyl acetate, ethyl acetate), ethers (e.g.dimethyl ether, diethyl ether, tetrahydrofuran, dioxane, monoglyme,diglyme, aromatic hydrocarbons (e.g. benzene, toluene, xylene),pyridine, dimethylformamide and suitable mixtures of two or more ofthese solvents. Catalysts for the catalytic reaction include palladium,platinum, rhodium and Raney nickel. Addition of a trace amount of aceticacid, trifluoroacetic acid, hydrochloric acid or sulfuric acid can allowthe reaction to proceed advantageously.

The method for production of the compound (I-1) is selected according tothe nature of --COOR¹ and --COOR² ; when --COOR¹ and --COOR² arecarboxyl groups esterified with methyl, ethyl, propyl, butyl, sec-butyl,phenyl or substituted phenyl group, the method A or the method B-1 isapplied advantageously; when --COOR¹ and --COOR² are carboxyl groupsesterified with iso-propyl or tert-butyl group, the method B-2 isapplied advantageously; and when --COOR¹ and --COOR² are carboxyl groupsesterified with benzyl or a substituted benzyl group, the method B-1 orthe method C is applied advantageously. When --COOR¹ and --COOR² aredifferent from each other, the methods A, B-1, B-2 and C may be combinedappropriately.

In the following the method for production of the starting compound (II)is explained.

The compound (II) wherein the ring A is a pyrrole ring, can be produced,for example, by the following processes. ##STR7##

In the reaction formulas described above, X, Y and R³ are the same asdescribed before; R^(a), R^(b) and R^(c) are independently a hydrogenatom, a fluorine atom or an alkyl group (the same as those representedby R described before); R⁴ is a cyano group or an esterified carboxylgroup represented by the formula --COOR⁶ ; A is a hydrogen atom or ahalogen atom (e.g. fluorine atom, chlorine atom, bromine atom, iodineatom); B is a halogen atom (e.g. chlorine atom, bromine atom, iodineatom) or an eliminable group which may be easily derived from hydroxygroup (e.g. methanesulfonyloxy group, benzenesulfonyloxy group,p-toluenesulfonyloxy group, trifluoromethanesulfonyloxy group); and m is0, 1 or 2. R⁶ in the esterified carboxyl group represented by theformula --COOR⁶ is exemplified by an alkyl group having 1 to 4 carbonatom(s) (e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,tert-butyl, etc.), phenyl or substituted phenyl group (p-nitrophenyl,p-methoxyphenyl, etc.), and benzyl or substituted benzyl (e.g.p-nitrobenzyl, p-methoxybenzyl, etc.).

The compound (V) may be dehydrogenated on the possible position betweenthe two adjacent carbons and form an unsaturated bond.

In the following the reaction processes described above are explained indetail.

Process 1

The compound (V) and the compound (VI) are subjected to condensation andthe resulting product is subjected to reduction to give the compound(VII).

For the condensation, a known reaction (e.g. aldol reaction, Reformatskyreaction, Witrig reaction, etc.) is employable, and for the reduction,usually a catalytic reduction under hydrogen atmosphere in the presenceof a catalyst (e.g. nickel, palladium, platinum, rhodium) isadvantageously employed.

In the condensation by aldol reaction, the employable base catalystsinclude metal byrdoxides such as sodium hydroxide, potassium hydroxide,lithium hydroxide and barium hydroxide, metal alkoxides such as sodiummethoxide, sodium ethoxide and potassium tert-butoxide, metal amidessuch as sodium amide and lithium diisopropylamide, metal hydrides suchas sodium hydride and potassium hydride, organic metal compounds such asphenyllithium and butyllithium and amines such as triethylamine,pyridine, α-, β- or γ-picoline, 2,6-lutidine, 4-dimethylaminopyridine,4-(1-pyrrolidinyllpyridine, dimethylaniline and diethylaniline; theemployable acid catalysts include mineral acids such as hydrochloricacid, sulfuric acid, nitric acid, phosphoric acid and boric acid, andorganic acids such as oxalic acid, tartaric acid, acetic acid,trifluoroacetic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and camphorsulfonic acid. The condensation can beconducted according to the known method [Ei-Ichi Negishi,Organometallics in Organic Synthesis, vol. 1, John Wiley & Sons, NewYork Chickester, Brisbane, Toronto (1980)] which comprises converting aketone form into the silylenolether form which is then subjected tocondensation with an aldehvde or an equivalent in the presence of aLewis acid [e.g. anhydrous zinc chloride, anhydrous aluminum chloride(AlCl₃), anhydrous ferric chloride, titanium tetrachloride (TiCl₄), tintetrachloride (SnCl₄), antimony pentachloride, cobalt chloride, cupricghloride, boron trifluoride ethyl ether complex, etc.], or converting aketone form into the enolate by treating the ketone form with a metaltriflate (e.g. dialkyl boron tin (II) triflate) in the presence ofamines (e.g. triethylamine, pyridine, α-, β- or γ-picoline,2,6-lutidine, 4-dimethylaminopyridine, 4-(1-pyrrolidinyi)pyridine,dimethylaniline, diethylaniline) followed by subjecting the enolate tocondensation with an aldehyde or an equivalent. The condensation isconducted in a suitable solvent at a temperature ranging from -100° C.to the boiling point of the solvent, preferably ranging from -78° to100° C., for 1 minute to 3 days. Solvents employable for the reactioninclude water, liquid ammonia, alcohols (e.g. methanol, ethanol,propanol, isopropanol, butyl alcohol, sec-butyl alcohol, tert-butylalcohol, ethylene glycol, methoxyethanol, ethoxyethanol), ethers (e.g.dimethyl ether, diethyl ether, tetrahydrofuran, dioxane, monoglyme,diglyme), halogenated hydrocarbons (e.g. dichloromethane, chloroform,carbon tetrachloride), aliphatic hydrocarbons (e.g. pentane, hexane,heptane), aromatic hydrocarbons (e.g. benzene, toluene, xylene),dimethylformamide, dimethylsulfoxide, hexamethylphospholamide,sulfolane, and the suitable mixtures thereof. In the condensation byWittig reaction, the employable reagents include metal hydroxides suchas sodium hydroxide, potassium hydroxide, lithium hydroxide and bariumhydroxide, metal alkoxides such as sodium meshoxide, sodium ethoxide andpotassium tert-butoxide, metal amides such as sodium amide and lithiumdiisopropylamide, metal hydrides such as sodium hydride and potassiumhydride, organic metal compounds such as phenyllithium and butyllithium,and amines such as triethylamine, pyridine, α-, β-, or γ-picoline,2,6-1utidine, 4-dimethylaminopyridine, 4-(1-pyrrolidinyl)pyridine,dimethylaniline and diethylaniline. The reaction is conducted in asuitable solvent at a temperature ranging from -20° C. to the boilingpoint of the solvent used, preferably ranging from 0° to 150° C., for 1minute to 10 days. The solvents employable for the reaction includeliquid ammonia, alcohols (e.g. methanol, ethanol, propanol, isopropanol,butyl alcohol, sec-butyl alcohol, tert-buty1 alcohol, ethylene glycol,mehoxyethanol, ethoxyethanol), ethers (e.g. dimethyl ether, diethylether, tetrahydrofuran, dioxane, monoglyme, diglyme, aliphatichydrocarbons (e.g. pentane, hexane, heptane), aromatic hydrocarbons(e.g. benzene, toluene, xylene), dimethylformamide, dimethylsulfoxide,hexamethylphospholamide, sulfolane and the suitable mixtures thereof.

The condensation can also be conducted by using a Reformatsky reaction.The reagents employable for the Reformatsky reaction include zinc,magnesium, aluminum and tin, and the reaction is conducted in a suitablesolvent at a temperature ranging from -20° C. to the boiling point ofthe solvent used, preferably ranging from 0° to 150° C., for 30 minutesto 3 days. The solvents employable for the reaction include ethers (e.g.dimethyl ether, diethyl ether, tetrahydrofuran, dioxane, monoglyme,diglyme), aliphatic hydrocarbons (e.g. pentane, hexane, heptane),aromatic hydrocarbons (e.g. benzene, toluene, xylene) and the suitablemixtures thereof.

The reaction conditions for the catalytic reduction are the same asthose for the deesterification at the --COOR¹ and --COOR² of thecompound (III) (method C).

The starting materials (V) and (VI) can be obtained easily according tothe known methods described in the literature. [B. Neises et al., Angew.Chem. Int. Ed. Engl., 17, 522 (1978)].

Process 2

This is the process whereby an eliminable functional group B isintroduced into the active methylene (the α-position of the carbonicacid ester) of the compound (VIII): it can be conducted easily by usingknown reagents according to a per se known method.

Process 3

The compound (VIII) obtained in the Process 2 is subjected tocondensation with matononitrile or a cyanoacetic acid ester [NC--CH₂COOR⁶ ; R⁶ is the same as described above] under a basic condition, togive the compound (IX). The employable bases, solvents and reactionconditions are in accordance with the known methods.

Process 4

The compound (IX), when treated with guanidine, can react at the cyanogroup or the ester residue followed by ring closure to form newly apyrrolopyrimidine ring. Ring closure under a basic condition allows thereaction to proceed advantageously. The employable bases include metalalkoxides such as sodium mothoxide, sodium ethoxide and potassiumtert-butoxide. The employable solvents for the reaction includemethanol, ethanol, propanol, tert-butyl alcohol, dimethylsulfoxide andhexamethylphospholamide. The reaction temperature ranges from 0° to 150°C., preferably from 20° to 100° C. The reaction time ranges from 1 to 48hours.

Process 5

The compound (IV-1: Y═NH₂ or OH) obtained in the Process 4 can beconverted into the compound (II-1: Y═NH₂ or OH) by subjecting the esterresidue [--COOR³ ] to the deesterification used in the preparation ofthe compound

Process 6

The compound (II-1: Y═OH) obtained in the Process 5 is subjected toreduction to give the compound (II-2): Y═H). The conditions for thereduction are per se known, and reduction by a metal hydride (e.g.borane, alane or ate complexes thereof) is employed advantageously.

The Process 5 and the Process 6 may be conducted in the reverse order.Namely, in the Process 7 the compound (IV-1: Y═OH) is subjected toreduction similar to that in the Process 6 to give the compound (IV-2:Y═H), which is then subjected to deesterification in the Process 8 in asimilar manner as in the Process 5 to give the compound (II-2: Y═H).Either the deesterification or the reduction can be selected to beconducted in advance to the other according to the nature of thesubstituents in the compound (IV-1: Y═OH).

In the above Processes 6 and 8, the mixture containing the compounds(II-2) and (II-2') or the compounds (IV-2) and (IV-2') may be separated,or each of the compounds (II-2) and (II-2') or each of the compounds(IV-2) and (IV-2') is synthesized predominantly by selective reduction.

Among the compounds those represented by the formula (II-3: X═OH)##STR8## wherein R and n mean the same as described before, can beobtained also by the following processes. ##STR9##

In the Processes described-above, R, Ra, Y and n mean the same asdescribed before and Z means the formula RCH₂ CO-- wherein R means thesame as described above, the formula ##STR10## wherein L is phenyl,butyl or cyclohexyl, and R and n mean the same as described above, orthe formula ##STR11## wherein M is ethyl or phenyl, and R and n mean thesame as described above. It is preferable that Y is hydrogen.

In the following these Processes are explained.

Process 9

The compound (X) [T. Kondo et al., Chemistry Letters, 419 (1983)] and apara-substituted benzoic acid ester derivative (XI) are subjected tocondensation (aldo reaction, Wittig reaction) followed by catalyticreduction under hydrogen atmosphere, to give the compound (XII). For thecondensation are applicable the reaction conditions, the reactionsolvents, the reaction temperatures and the reagents used in the ProcessI. For the catalytic reduction under hydrogen atmosphere are applicablethe conditions used in the deesterification of --COOR¹ and --COOR² ofthe compound (III).

Process 10

Treatment of the compound (XII) under acidic conditions can eliminatethe protection of the isopropyloxymethyl group at the 3-position to givethe compound (XIII). The conditions, solvents and temperatures used indeesterification of --COOR¹ and --COOR² of the compound (III) (themethod B-1 and the method B-2) are employable for the reaction.

Process 11

The compound (XIII) obtained in the Process 10 is subjected todehydrogenation by a per se known method, to be easily converted intothe compound (IV-3: Y═H).

Process 12

The compound (IV-3: Y═H) obtained in the Process 11 can be convertedinto the compound (II-3) by deesterification. The conditions, solventsand temperatures described in detail for the deesterification of --COOR¹and --COOR² of the compound (III) (the methods A, B-1, B-2 and C) areemployable for the reaction. The processes 10 to 12 may be conducted inany order with the formation of the respective products, and finally thedesired compound (II-3) is obtained. The order is determined suitablyaccording to the nature of the substituents of the compounds (XII),(XIII) and (IV-3). The compound (II-3) thus obtained can be converted,if necessary, into the compound (II-2) by a known substituent-convertingreaction on the pyrimidine ring reported in the literature. [ProteinNucleic acid Enzyme Extra issue, Chemical synthesis of nucteic acids,Kyoritsu Shuppan (1968)].

The compounds other than the compound (II-3), wherein X is hydroxyl canbe also converted into the corresponding compounds wherein X is amino bythe above-mentioned substituent-converting reaction.

The reactions, reagents and reaction conditions used in the Processes 1to 12 and in the production of the starting compounds (V) and (XIII) areknown and explained in detail in the following literature. [J. F. W.Mcomine, Protective Groups in Organic Chemistry, Plenum Press, Londonand New York (1973)], [Pine, Hendrikson, Hammond, Organic Chemistry (4thedition) [I]-[II], Hirokawa Shoten (1982)], and [M. Fieser and L.Fieser, Reagents for Organic Synthesis vol. 1-10, Wiley-Interscience,New York, London, Sydney and Toronto (1969-1982)].

The intermediates of the compounds of this invention and the compounds(I) of this invention can be isolated from the reaction mixtures by theconventional means for separation and purification, such asconcentration, extraction with solvent, chromatography andrecrystallization.

The compounds (I), (II) and (IV) of this invention may form salts. Suchsalts are produced by the known methods, and exemplified by the salts ofpharmaceutically acceptable bases or acids and quaternary salts. Saltsof bases include salts of alkali metals, alkali earth metals, non-toxicmetals, ammonium and substituted ammonium, such as sodium, potassium,lithium, calcium, magnesium, aluminum, zinc, ammonium,trimethylammonium, triethylammonium,. triethanolammonium, pyridinium andsubstituted pyridinium. Salts of acids include salts of mineral acidssuch as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acidand boric acid, and salts of organic acids such as oxalic acid, tartaricacid, acetic acid, trifluoroacetic acid, methanesulfonic acid andcamphorsulfonic acid. Quaternary salts include salts of methyl bromide,methyl iodide, methyl methanesulfonate, methyl benzensulfonate andmethyl p-toluenesulfonate. Also, the compounds (I), (II) and (IV) mayform zwitterion.

As the compounds (I) of this invention, the following compounds areexemplified:

DiethylN-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]-pyrimidin-5-yl)propyl]benzoyl]-L-glutamate,

N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamicacid,

DiethylN-[4-[3-(2,4-diamino-6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5yl)propyl]benzoyl]-L-glutamate,

N-[4-[3-(2,4-diamino-6-hydroxy-7H-pyrrolo[2,3-d]-pyrimidin-5-yl)propyl]benzoyl]-L-glutamicacid,

Diethyl N-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5yl)propybenzoyl]-L-glutamate,

N-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamicacid,

Diethyl N-[4-[3-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin5-yl)propyl]benzoyl]-L-glutamate,

DiethylN-[4-[2-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl-L-glutamate,

DiethylN-[4-[3-(2,4-diamino-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamate,

N-[4-[3-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamicacid,

N-[4-[2-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid,

DiethylN-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]-L-glutamate,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]-L-glutamicacid,

DiethylN-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-methyl-propyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-ethylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-5]-ethylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-1vinylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5yl)-2-vinylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-1-allylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-2-allylpropyt]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-(propen-1-yl)propyl]benzoyl-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-(propen-1-yl)-propyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-1-ethynylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-2ethynylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-1-propagylpropyl]benzoyl-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-2-propagylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-1-(propyn-1-propyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-1-(propyn-1-yl)propyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-7H-pyrro[2,3-d]pyrimidin-5-yl)-2-(propyn-1-yl)-propyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-2-methylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-1-ethylpropyl]benzoyl]-L-glutamticacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-2-ethylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-1-vinylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-]pyrimidin-5-yl)-2-vinylpropyl]benzoyl]-L-glutamaticacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-1-allylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-2-allylpropyl]benzoyl]-L-glutamaticacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-1-(propen-1-yl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-2-(propen-1-yl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-1-ethylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-2-ethylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-1-propagylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-2-propagylpropyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-1-(propyn-1-yl)propyl]benzoyl]-L-glutamicacid,

N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrro[2,3-d]pyrimidin-5-yl)-2-(propyn-1-yl)propyl]benzoyl]-L-glutamicacid.

Effects

The compounds (I) of this invention show excellent antitumor effects inmouse tumor cell strains (P388, L1210, L5178Y, B16 melanoma, MethA,Lewis Lung Carcinoma, S180 sarcoma, Ehrlich Carcinoma, Colon38) andhuman tumor cell strains (HL60, KB, Lu65), decrease the tumors carriedby warm-blooded animals [e.g. melanoma, sarcoma, mastocytoma, carcinoma,neoptasia, etc.] and prolong the life-span of tumor-carryingwarm-blooded animals.

In the following are described the results indicating the pharmaceuticaleffects of the compounds (I) of this invention.

The cell growth inhibiting effect (IC₅₀) of the compounds obtained inthe Workina Examples described below in KB cells was determined by thefollowing method.

Human nasopharyngeal cancer KB cells (1×10⁴ cells/ml) prepared accordingto a conventional method were inoculated into each well of the96-microwell plate (0.1 ml in a well) and subjected to standing cultureat 37° C. under 5% CO₂ for 24 hours. To this was added a solution of oneof the compounds obtained in the Working Examples in 10% MEM (NissuiPharmaceutical Co. Ltd.), and subjected again to standing culture at 37°C. under 5% CO₂ for 72 hours. Then the culture was piperted out, andanother 0.1 ml of the solution of MTT (Dojindo Laboratories) in 10% MEM(1.0 mg/ml) was added and incubated at 37° C. for 4 hours. Then 0.1 mlof the 10% SDS solution (Wako Pure Chemicals) was added and incubated at37° C. for further 24 hours. The absorbance at 590 nm was measured andthe IC₅₀ value of the compound was defined as the concentration of thecompound required to decrease the number of cells in the untreatedcontrol group by 50%. The results obtained are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Test compound        IC.sub.50 (μg/ml)                                     ______________________________________                                        Compound of Working Example 4                                                                      0.0003                                                   Compound of Working Example 6                                                                      0.08                                                     Compound of Working Example 16                                                                     0.0006                                                   ______________________________________                                    

In addition, the following are described the results indicating thepharmaceutical effects of the compounds (I) of this invention.

The cell growth inhibiting effect (IC₅₀) of the compound obtained in theWorking Example 14 described below in HL-60 and HEL cells was determinedby the following method.

(1) Human Leukemia cells HL-60 (2×10⁵ cetls/ml) were suspended in theGIT culture medium (Wako Pure Chemicals) containing the compound of thisinvention and 0.2 ml of the suspension was inoculated into each well ofthe 96-microwell plate. After standing culture at 37° C. under 5% CO₂for 68 hours, 1 μCi of [³ H]-thymidine (5 Ci/mmol) was added and themixture was incubated for 4 further hours. For measurement of theincorporation of thymidine into the cells, the acid-insoluble fractionwas collected on a glass filter, and the radioactivity of the fractionwas measured by a liquid scintillation counter. The IC₅₀ value of thecompound was defined as the concentration of the compound required todecrease the radioactivity incorporated into the cells in the untreatedcontrol group by 50%.

(2) Human fetal normal lung fibroblasts HEL (1×10⁴ cells/ml) weresuspended in the MEM culture medium (Nippon Flow Laboratories) and 0.1ml of the suspension was inoculated into each well of the 96-microwellplate. After standing culture at 37° C. under 5% CO₂ for 24 hours, MEMculture medium containing the compound of this invention was added andthe mixture was incubated for further 72 hours. The medium was replacedby the medium containing 1 μg/ml of MTT (Dojindo Laboratories), to whichwas added 10% SDS (Wako Pure Chemicals), and incubated overnight.Absorbance at 590 nm was measured by he Multiscan (Titertec Co.). TheIC₅₀ value was determined by comparing the absorbance in the untreatedcontrel group. The results obtained are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                             HL-60    HEL                                             Test compound        (μg/ml)                                                                             (μg/ml)                                      ______________________________________                                        Compound of Working Example 14                                                                     0.04     >20.0                                           ______________________________________                                    

As shown by the above-mentioned results, the compounds (I) are excellentin inhibition of cell growth of KB and HL-60, while they do not exert atoxicity against HEL. The compounds (I) of this invention and the saltsthereof are of low-toxicity, having remarkable antitumor effect.Therefore, the preparations containing the compound (I) or salts thereofcan be employed as antitumor agents for the treatment of tumors inwarm-blooded animals, particularly mammals (e.g. mouse, rat, cat,, dog,rabbit, etc.).

The compounds (I) and salts thereof, when used as antitumor agents, canbe administered orally and parenterally as they are or in the forms ofpowders, granules, tablets, capsules, suppositories and injections,which are prepared according to the conventional methods usingpharmaceutically acceptable excipients, vehicles, and diluents. The dosevaries according to the animals, diseases, symptoms, compounds andadministration routes; for example, the daily dose is about 2.0 to 100mg of a compound of this invention per kg of body weight of awarm-blooded animal described above for oral administration, and about1.0 to 50 mg/kg for parenteral administration. Injections may beadministered intramuscularly, intraperitoneally, subsutaneously orintravenously.

The preparations are produced by the per se known processes. For theabove-mentioned oral preparations, for example, tablets are produced bysuitable combination with a binder (e.g. hydroxypropylcellulose,hydroxypropytmethylcellulose, macrogol, etc.), a disintegrator (e.g.starch, calcium carboxylmethylcellulose, etc.) and a lubricant (e.g.magnesium stearate, talc, etc.).

As parenteral preparations, for example, injections are produced bysuitable combination with an agent to provide isotonicity (e.g. glucose,D-sorbitol, D-mannitol, sodium chloride, etc.), an antiseptic (e.g.benzyt alcohol, chlorobutanol, methyl p-hyrdoxybenzoate, propylp-hydroxybenzoate, etc.) and a buffer (e.g. phosphate buffer, sodiumacetate buffer, etc.).

An example process for production of tablets comprises mixing about 1.0to 25 mg of the compound of this invention, 100 to 500 mg of lactose,about 50 to 100 mg of corn starch and about 5 to 20 mg ofhydroxypropylcellulose for preparation of a tablet by a conventionalmeans, granulating, mixing with corn starch and magnesium stearate andtabletting, so that tablets each weighing about 100 to 500 mg with thediameter of about 3 to 10 mm are obtained. The tablets may be coatedwith a mixture of acetone and ethanol, the mixture containinghydroxypropylmethylceilulose phthalate (about 10 to 20 mg per tablet)and castor oil (0.5 to 2 mg) at a concentration of about 5 to 10%, togive enteric coated tablets.

An example process for injectable preparation comprises dissolving about2.0 to 50 mg of a sodium salt of the compound of this invention in about2 ml of physiological saline for preparation of an ampoule, sealing theresultant solution in an ampoule and sterilizing the ampoule at 110° C.for about 30 minutes or adding about 2 ml of sterile distilled watercontaining about 10 to 40 mg of mannitol or sorbitol into the ampoule,freeze-drying and sealing the ampoule. For use of the freeze-driedcomoound for subcutaneous, intravneous or intramuscular injection, theampoule is opened and the content is dissolved in, for example,physioloaical saline so that the concentration of the compound may beabout 1.0 to 25 mg/ml.

The following Reference Examples and Working Examples will explain thepresent invention more concretely.

Reference Example 1

Production of methyl 5-[4-(tert-butoxycarbonyl)phenyl]pentanoate:

Under an atmosphere of argon, potassium (25 g) was added to driedtert-butyl alcohol (820 ml), which was refluxed by heating to bedissolved completely. The solution was cooled to 20° C., to which ether(300 ml) was added and then a solution of methyl crotonate (63.93 g) andtert-butyl 4-formylbenzoate (71.0 g) in tert-butyl alcohol-ether (2:1,300 ml) was added slowly while the inner temperature was kept at 10° C.After stirring at the same temperature for 2 hours, 1N potassiumhydrogen sulfate in water (750 ml) was added with cooling so that the pHwas adjusted to 4. The solution was extracted with ether, washed withwater and then with saturated sodium chloride solution and subjected toevaporation of the solvent under reduced pressure. The resultant residuewas dissolved in ethyl acetate (100 ml), to which 5%Pd-C (15 g:Engelhard Co. Ltd.) was added and stirred vigorously under hydrogenpressure of 4 kg/cm² at room temperature for 3 hours. The catalyst wasfiltered off, the solvent was evaporated under reduced pressure, to theresidue were added dried methanol (200 ml),4-(N,N-dimethylamino)pyridine (30 mg) and dichtoromethane (250 ml), andthen a solution of 1,3-dicyclohexylcarbodiimide (132 g) indichloromethane (250 ml) was slowly added dropwise at 0° C. Afterstirring at room temperature for 18 hours, the mixture was cooled to 0°C.; which acetic acid (30 ml) was added and the mixture was stirred at0° C. for 30 minutes and then at room temperature for 30 minutes. Theresultant precipitate was filtrated off, the filtrate was concentratedto dryness under reduced pressure, to the residue was added ethylacetate (100 ml) and after keeping at 0° C. for 2 hours, the resultantprecipitate was again filtrated off. The filtrate was concentrated underreduced pressure and the residue was purified by column chromatography(carrier; silica gel, 100 g, developing solvent; ether:hexane=1:15→1:5), to give the object compound (59.7 g). melting point(Bp) 145°-155° C./0.2-0.3 mmHg

IR (Neat): 2980, 2950, 1740, 1712, 1605 cm⁻¹ :

¹ H-NMR (CDCl₃) δ: 1.40-1.75 (4H,m), 1.55 (9H, s), 2.15-2.45 (2H,m),2.50-2.75 (2H,m), 3.62 (3H s), 7.16 (2H,d,J=8 Hz), 7.85 (H,d,J=8 Hz).

Reference Example 2

Production of methyl 5-[4-(tert-butoxycarbonyl)phenyt]-2-iodopentanoate:

Under an atmosphere of argon, to a solution of diisopropylamine (2.48 g)in tetrahydrofuran (100 ml) was added a solution of butyllithium (24.5mmol) in hexane (15.3 ml) at 0° C. and stirred for 10 minutes; to this asolution of the compound (6.53 g) obtained in the Reference Example 1 intetrahydrofuran (50 ml) was added dropwise at -78° C. over 30 minutes.After stirring for 30 minutes, a solution of iodine (5.66 g) intetrahydrofuran (30 ml) was added and stirred for further 20 minutes.The temperature of the solution was brought up to 0° C. over 30 minutes,1N potassium hydrogen sulfate in water (30 ml) was added dropwise, andthe solution was extracted with ether after adjustment to pH 4. Theorganic layer was washed with 1N potassium carbonate in water and thenwith saturated sodium chloride solution, and dried with anhydrousmagnesium sulfate. The residue obtained by evaporation of the solventunder reduced pressure was purified by column chromatography(ether-hexane, 1:9), to give the object compound (4.736 g).

IR (Neat): 2990, 2905, 1744, 1718, 1612 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.45-1.80 (2H,m), 1.58 (9H,s), 1.80-2.16 (2H,m), 2.69(2H,t,J=7 Hz), 3.72 (2H,s), 4.30 (1H,t,J=7 Hz), 7.20 (2H,s,J=8 Hz), 7.90(2H,d,J=8 Hz).

Reference Example 3

Preparation of methyl5-[4-(tert-butoxycarbonyl)phenyl]-2-(dicyanomethyl)pentanoate:

To a suspension of sodium hydride (1.356 g) in dimethylsulfoxide (8 ml)was added a solution of malononitrile (3.37 g) in dimethylsulfoxide (8ml) under cooling with water, and stirred for 15 minutes. To thissolution was added dropwise a solution of the compound (4.736 g)obtained in the Reference Example 2 in dimethylsulfoxide (12 ml) andstirred at room temperature for 1 hour, to this 45 ml of 1N potassiumhydrogen sulfate in water was added at 0° C., followed by extractionwith ether. The ether layer was washed with water and dried withanhydrous magnesium sulfate, followed by evaporation of the solventunder reduced pressure. The residue was purified by columnchromatography (carrier; silica gel, 200 g, developing solvent; ethylacetate: hexane=1:5), to give the object compound (3.33 g).

IR (Neat): 2970, 2930, 2252 1740, 1713, 1608 cm³¹ 1

¹ H-NMR (CDCl₃) δ: 1.60-2.05 (4H,m), 1.48 (9H,s), 2.70 (2H,brt,J=7 Hz),2.90-3.15 (1H,m), 3.82 (3H,s), 4.04 (1H,d,J=7 Hz), 7.20 (2H, d,J=8 Hz),7.92 (2H,d,J=8 Hz).

Reference Example 4

Production of methyl4-[3-(2-amino-7-benzyl-3isopropyloxymethyl-4(3H)-oxopyrrolo[2,3-d]pyrimidin-5-yl)-1-oxo-2-propenyl]benzoate:

2-Amino-7-benzyl-3-isopropyloxymehtyl-4(3H)-oxopyrrolo[2,3-d]pyrimidine-5-carbaldehyde(1.7 g) was suspended in a methanol-tetrahydrofuran mixture (10:1, 33ml), to which a solution of sodium methylate in methanol (equivalent to6.25 mM, 3.75 ml) was added to dissolve. Then4-methoxycarbonylacetophenone (2.23 g) was added and stirred at roomtemperature for 15 hours. The precipitate was collected by filtration,washed with a small amount of methanol and ether and dried, to give theobject compound (2.02 g) as yellow needles.

IR (KBr): 3480, 3350, 1710, 1680, 1620, 1550 1375, 1280, 1210, 1110,1060, 775. cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.23 (6H,d,J=6 Hz), 3.93 (3H,s), 3.80-4.07 (1H,m),5.15 (2H,s), 5.63 (2H,s), 6.92 (1H,s), 7.10-7.40 (5H, m), 7.73(1H,d,J=15 Hz), 8.13 (4H,s), 8.60 (1H,d,J-15 Hz).

Reference Example 5

Production of methyl4-[3-(2-amino-3-isopropyloxymethyl-4(3H)-oxo-5,6-dihydropyrrolo-[2,3-d]pyrimidin-5-yl)propyl]benzoate:

The compound (2.01 g) obtained in Reference Example 4 was dissolved in amethanol-tetrahydrofuran mixture (3:4, 350 ml), to which 1N hydrochloricacid (8 ml) and 10% Pd-C (4 g, manufactured by Engelhard Co. Ltd.) wereadded, and subjected to catalytic reduction under an atmosphere ofhydrogen for 48 hours. The catalyst was filtrated off, the filtrate wasneutralized, the solvent was evaporated off under reduced pressure andthe residue was isolated and purified by column chromatography on silicagel (carrier; 100 g, developing solvent; chloroform containing 2-4% ofethanol), to give the object compound (0.68 g) as a colorless powder.

IR (KBr): 3210, 2980, 1725, 1625, 1580, 1510, 1435, 1275, 1175, 1100,1060 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.17(3H,d,J=6 Hz), 1.19 (3H,d,J=6 Hz), 1.50-2.13(4H,m), 2.70 (2H,t,J=7.5 Hz), 3.07-3.77 (3H,m), 3.80-4.60 (1H,m), 3.87(3H,s), 5.03 and 5.57 (2H,ABq), 7.21 (2H,d,J=7.5 Hz), 7.91 (2H,d,J=7.5Hz).

Reference Example 6

Production of menhyl 4-[3-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoate:

The compound (0.66 g) obtained in the Reference Example 5 was dissolvedin dried tetrahydrofuran (31.5 ml), to which 0.21N hydrogen bromide indichloromethane (78.3 ml) was added and stirred at room temperature for20 hours. Then 3 volumes of n-hexane was added and the resultantprecipitate was collected by filtration, to give the dihydrobomide ofthe object compound (0.59 g) as a colorless powder.

IR (KBr): 3290, 3030, 2950, 1720, 1690, 1680 1620, 1480, 1350, 1275,1100, 1035, 760 cm⁻¹.

¹ H-NMR (DMSO-d₆) δ: 1.40-1.83 (4H,broad), 2.65 (2H,t,J=7.5 Hz),3.07-3.37 (2H,m), 3.50-3.77 (1H),m), 3.82 (3H,s), 7.33 (2H,d,J=7.5 Hz),7.86 (2H,d,J=7.5 Hz).

Reference Example 7

Production of diethylN-[4-[3-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamate:

The compound (1.47 g) obtained in the Reference Example 6 was suspendedin tetrahydrofuran (60 ml), to which 0.1N sodium hydroxide in water (20ml) was added and stirred at room temperature for 21 hours. Then thesolution was neutralized with 0.1N hydrochloric acid (60 ml) andconcentrated to dryness under reduced pressure. The residue wassuspended in dried dimethylformamide (112.5 ml), to which diethylL-glutamate hydrochloride (2.88 g), diphenylphosphoryl azide (1.295 ml)and triethylamide (2.52 ml) were added, brought back to the roomtemperature and stirring was continued for 63 hours. The resultingprecipitate was filtrated off, and the filtrate was concentrated todryness under reduced pressure. The residue was subjected toseparation-purification with column chromatography on silica col(carrier; 100 g, developing solvent; chloroform containing 6.9%ammonia-containing ethanol, 1:20-1:10), to give the object compound(1.12 g) as a colorless powder.

IR (KBr): 3330, 2930, 1740, 1670, 1640, 1570, 1540, 1440, 1375, 1300,1200, 1095, 1020 cm⁻¹.

¹ H-NMR (CDCl₃ -CD₃ OD) δ: 1.20 (3H,t,J=7.5 Hz), 1.27 (3H,t,J=7.5 Hz),1.47-1.83 (4H,m), 2.0-2.36 (2H,m), 2.37-2.50 (2H,m), 2.67 (2H,t,J=7.5Hz), 3.10-3.37 (2H,m), 3.53-3.80 (1H,m), 3.96-4.33 (4H,q×2,J=7.5 Hz),4.60-4.87 (1H,m), 7.25 (2H,d, J=9 Hz), 7.75 (2H,d,J=9 Hz).

Reference Example 8

Production of methyl4-[3-(2-amino-7-benzyl-3-isopropyloxymethyl-4(3H)-oxopyrrolo[2,3-d]pyrimidin-5-yl)-1-oxo-2-propenyl]benzoate:

2-Amino-7-benzyl-3-isopropyloxymethyl-4(3H)-oxopyrrolo[2,3-d]pyrimidine-5-carbaldehyde(1.7 g) was suspended in a methanol-tetrahydrofuran mixture (10:1, 33ml), to which a solution of sodium methylate in methanol (equivalent to6.25 mM, 3.75 ml) was added to dissolve. Then4-methoxycarbonylacetophenone (2.23 g) was added and stirred at roomtemperature for 15 hours. The precipitate was collected by filtration,washed with s small amount of methanol and ether, and dried, to give theobject compound (2.02 as yellow needles.

IR (KBr): 3480, 3350, 1710, 1680, 1620, 1550, 1375, 1280, 1210, 1110,1060, 775 cm⁻¹

¹ H-NMR (CDCl₃) δ: 1.23 (6H,d,J=6 Hz), 3.93 (3H,s), 3.80-4.07 (1H,m),5.15 (2H,s), 5.63 (2H,s), 6.92 (1H,s), 7.10-7.40 (5H,m), 7.73 (1H,d,J=15Hz), 8.13 (4H,s), 8.60 (1H,d,J=15 Hz).

Reference Example 9

Production of methyl4-[3-(2-amino-3-isopropyloxymethyl-4(3H)-oxo-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoate:

The compound (2.01 g) obtained in Reference Example 8 was dissolved in amethanol-tetrahydrofuran mixture (3:4, 350 ml), to which 1N hydrochloricacid (8 ml) and 10% Pd-C (4 g, manufactured by Engelhard Co. Ltd.) wereadded, and subjected to catalytic reduction under an atmosphere ofhydrogen for 48 hgurs. The catalyst was filtrated off, the filtrate wasneutralized, the solvent was evaporated off under reduced pressure, andthe residue was isolated and purified by column chromatography on silicagel (carrier; 100 g) (developing solvent: chloroform containing 2-4% ofethanol), to give the object compound (0.68 g) as a colorless powder.

IR (KBr): 3210, 2980, 1725, 1625, 1580, 1510, 1435, 1275, 1175, 1100,1060 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.17 (3H,d,J=6 Hz), 1.19(3H,d,J=6Hz), 1.50-2.13(4H,m), 2.70 (2H,t,J=7.5 Hz), 3.07-3.77 (3H,m), 3.80-4.06 (1H,m), 3.87(3H,s), 5.03 and 5.57 (2H,ABq), 7.21 (2H,d,J=7.5 Hz), 7.91 (2H,d,J=7.5Hz).

Reference Example 10

Production of methyl4-[3-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoate:

The compound (0.66 g) obtained in the Reference Example 9 was dissolvedin dried tetrahydrofuran (31.5 ml), to which 0.21N hydrogen bromide acidin dichloromethane (78.3 ml) was added, and stirred at room temperaturefor 20 hours. Then 3 volumes of n-hexane was added and the resultantprecipitate was collected by filtration, to give the dihydrobromide ofthe object compound (0.59 g) as a colorless powder.

IR (KBr): 3290, 3030, 2950, 1720, 1690, 1680, 1620, 1480, 1350, 1275,1100, 1035, 760 cm⁻¹.

¹ H-NMR (DMSO-d₆) δ: 1.40-1.83 (4H, broad), 2.65 (2H,t,J=7.5 Hz),3.07-3.37 (2H,m), 3.50-3.77 (1H,m), 3.82 (3H,s), 7.33 (2H,d,J=7.5 Hz),7.86 (2H,d,J=7.5 Hz).

Reference Example 11

Production of methyl4-[2-(2-amino-7-benzyl-3-isopropyloxymethyl-4(3H)-oxopyrrolo[2,3-d]pyrimidin-5-yl)ethenyl]benzoate:

To the suspension of2-amino-7-benzyl-3-isopropyloxymethyl-4(3H)-oxopyrrolo[2,3-d]pyrimidine-5-carbaldehyde(2.04 g) in dried methanol (84 ml) was addedp-methoxycarbonylbenzyltriphenylphosphonium bromide (3.24 g) andstirred. Then a solution of sodium methylate in methanol (equivalent to6.6 mM on sodium basis) was added and stirred at room temperature for1.5 hours, to give yellow needles. The needles were collected byfiltration, washed with methanol and then with ether, and dried, to givethe object product (cis-form, 1.49 g). The mother liquor was purified bycolumn chromatography on silica gel (carrier: 100 g) (developingsolvent: ethyl acetate-hexane, 1:4-1:3), to give the cis-trans mixtureof the object compound (0.9 g) as yellow powders. cis-form;

IR (KBr): 33.40, 3220, 2980, 1715, 1690, 1625, 1600, 1530, 1430, 1280,1175, 1105, 1060, 995 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.20 (6H,d,J=6 Hz), 3.87 (3H,s), 3.80-4.07 (1H,m),5.14 (2H,s), 5.32 (2H,s), 5.60 (2H,s), 6.77 (1H,s), 7.10-7.37 (5H,m),7.43 (2H,s), 7.50 (2H,d,J=9 Hz), 7.95 (2H,d,J=9 Hz).

Reference Example 12

Production of methyl 4-[2-(2-amino-3-isopropyloxymethyl-4(3H)-oxo-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoate:

The compound (1.6 g) obtained in the Reference Example 11 was subjectedto the same reaction to that in the Reference Example 9, to give theobject compound (0.62 g).

¹ H-NMR (CDCl₃) δ: 1.17 (3H,d,J=6 Hz), 1.20 (3H,d,J=6 Hz), 1.47-2.0(1H,m), 2.10-2.43 (1H,m), 2.65 (2H,t,J=9 Hz), 2.97-3.60 (3H,m),3.73-4.07 (1H,m), 3.90 (3H,s), 4.47 (2H,s), 5.30 (1H,d,J=12 Hz), 5.60(1H,d,J=12 Hz), 7.13-7.50 (7H,m), 7.92 (1H,d,J=9 Hz)

Reference Example 13

Production of methyl4-[2-(2-amino-4-hydroxy-7H-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-y1)ethyl]benzoate:

The compound (1.25 g) obtained in the Reference Example 12 was subjectedto the same reaction to that in the Reference Example 10, to give theobject compound (0.5g).

IR (KBr): 3400, 3300, 2920, 1740, 1710, 1680, 1640, 1600, 1570, 1435,1310, 1280, 1110, 1020 cm⁻¹.

¹ H-NMR (DMSO-d₆ /D₂ O) δ: 1.53-2.27 (2H,m), 2.70 (2H,t,J=9 Hz),3.00-3.26 (2H,m) 3.47-3.63 (1H,m), 3.83 (3H,s), 7.35 (2H,d J=9 Hz), 7.85(2H,d,J=9 Hz),

Reference Example 14

Production of methyl 5-[4-(tert-butoxycarbonyl)phenyl]pentanoate:

Under an atmosphere of argon, potassium (25 g) was dissolved completelyin dried tert-butyl alcohol (820 ml) by refluxing by heating for 3hours. The solution was cooled to 20° C., to which was added ether (300ml) and then slowly a solution of methyl crotonate (63.93 g) andcert-butyl 4-formylbenzoate (71.0 g) in tert-butyt alcohol-ether mixture(2:1, 300 ml) while keeping the inner temperature at 10° C.

The mixture was stirred at the same temperature for 2 hours, and 1Npotassium hydrogen sulfate in water (750 ml) was added with cooling toadjust the pH to 4. After extraction with ether, the ether layer waswashed with saturated sodium chloride solution and the solvent wasevaporated off under reduced pressure. The residue was dissolved inethyl acetate (100 ml), to which was added 5% Pd-C (15 g: manufacturedby Engelhard Co. Ltd.), and stirred vigorously at room temperature underhydrogen pressure of 4 kg/cm² for 3 hours. The catalyst was filteredoff, the solvent was evaporated off under reduced pressure, and driedmethanol (200 ml), 4-(N,N-dimethylamino)pyridine (30 mg) anddichloromethane (250 ml) were added, to which a solution of1,3-dicyclohexylcarbodiimide (132 q) in dichloromethane (250 ml) wasadded slowly dropwise at 0° C. After stirring at room temperature for 18hours, the mixture was cooled to 0° C. Acetic acid (30 ml) was added andthe mixture stirred at 0° C. for 30 minutes and then at room temperaturefor 30 minutes. The resulting precipitate was filtered off, the filtratewas concentrated to dryness under reduced pressure, and to the residuewas added ethyl acetate (100 ml, which was left standing at 0° C. for 2hours, and the resulting precipitate was again filtered off. TheFiltrate was concentrated under reduced pressure, and the residue waspurified by column chromatography (carrier: silica gel, 500 g,ether-hexane, 1:15-1:5), to give the object compound (59.7 g).

Bp. 145°-155° C./0.2-0.3 mmHg.

IR (Neat): 2980, 2950, 1740, 1712, 1605 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.40-1.75 (4H,m), 1.55 (9H,s), 2.15-2.45 (2H,m),2.50-2.75 (2H,m), 3.62 (3H,s), 7.16 (2H,d,J=8 Hz), 7.85 (H,d,J=8 Hz).

Reference Example 15

Production of methyl 5-[4-(tert-butoxycarbnyl)phenyl]-2-iodopentanoate:

Under an atmosphere of argon, a solution of butyllithium (24.5 mmol) inhexane (15.3 ml) was added to a solution of diisopropylamine (2.48 g) intetrahydrofuran (100 ml) at 0° C. and stirred for 10 minutes. To theresultant solution was added a solution of the compound (6.53 g)obtained in the Reference Example 14 in tetrahydrofuran (50 ml) at -78°C. dropwise over 30 minutes. After stirring for 30 minutes a solution ofiodine (5.66 g) in tetrahydrofuran (30 ml) was added and stirred forfurther 20 minutes. The temoerature was increased to 0° C. over 30minutes, 30 ml of 1N potassium hydrogen sulfate in water was addeddropwise, and the pH was adjusted to 4, followed by extraction withether. The organic layer was washed with 1N potassium carbonate in waterand then with saturated sodium chloride solution, and dried withanhydrous magnesium sulfate. The solvent was evaporated off underreduced pressure, and the resultant residue was purified by columnchromatography (carrier: silica gel, 100 g, ether-hexane, 1:9), to givethe object compound (4.736 g).

IR (Neat): 2990, 2905, 1744, 1718, 1612 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.45-1.80 (2H,m), 1.58 (9H,s), 1.80-2.16 (2H m), 2.69(2H,t,J=7 Hz), 3.72 (2H,s), 4.30 (1H,t,J=7 Hz), 7.20(2H,d,J=8 Hz),7.90(2H d J=8 Hz)

Reference Example 16

Production of methyl5-[4-(tert-butoxycarbonyl)-phenyl]-2-(dicyanomethyl)pentanoate:

To a suspension of sodium hydride (1.356 g) in dimethylsulfoxide (8 ml)was added a solution of malononitrile (3.37 g) in dimethytsulfoxide (8ml) with ice-cooling and stirred for 15 minutes. To the solution wasadded a solution of the compound (4.736 g) obtained in the ReferenceExample 15 in dimethylsulfoxide (12 ml) dropwise, and the mixture wasstirred at room temperature for 1 hour. Then 45 ml of 1N potassiumhydrogen sulfate in water was added at 0° C., followed by extractionwith ether. The ether layer was washed with water and dried withanhydrous magnesium sulfate. The residue obtained by evaooration of thesolvent under reduced oressure was purified by column chromatography(carrier: silica gel, 200 g, ethyl acetate-hexane, 1:5), to give theobject compound (3.33 g).

IR (Neat): 29.70, 2930, 2252, 1740, 1713, 1608 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.60-2.05 (4H,m), 1.48 (9H,s), 2.70 (2H,brt,J=7 Hz),2.90-3.15 (1H,m), 3.82(3H,s), 4.04(1H,d,J=7 Hz), 7.20(2H,d,J=8 Hz),7.92(2H,d,J=8 Hz).

Reference Example 17

Production of ethyl 5-[4-(tert-butoxycarbonyl)-phenyl]hexanoate:

In a solution of tert-butyl 4-acetylbenzoate (19.90 g) in abenzene-ether-tetrahydrofuran mixture (3:3:2, 200 ml) was suspended zinc(11.81 g), to which ethyl 4-bromocrotonate (17.44 g) was added slowlywhile heating and stirring, and then iodine (about 20 mg) was added. Theresulting mixture was refluxed by heating on an oil bath (60°-70° C.)for 1 hour, then ethyl 4-bromocrotonate (3.00 g) was added, and themixture was further refluxed by heating for 15 minutes. After cooling toroom temperature, the reaction mixture was added to water (500 ml),adjusted to pH 4.9 by addition of acetic acid and extracted with ether.The extract was washed with 5% aqueous ammonia and dried with anhydrousmagnesium sulfate.

The residue obtained by evaporation of the solvent under reducedpressure was purified by column chromatography (carrier; silica gel, 300g, developing solvent; ethyl acetate-hexane=1:5), to give the objectcompound.

IR (Neat): 3480, 2975, 1720, 1700, 1550, 1505 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.20 (3H,t,J=7 Hz), 1.53 (12H,s), 2.64 (2H,d,J=7 Hz),2.67 (1H,brs),3.63 (3H,s), 4.08 (2H,q,J=7 Hz), 5.80 (1H,d,J=15 Hz), 6.80(1H,dt,J=15 Hz 7 Hz), 7.45 (2H,d,J=8 Hz), 7.90 (2H,d,J=8 Hz).

The entire amount of ethyl hexenate derivative (22.3 g) was dissolved inan ethanol-acetic acid mixture (20:1, 200 ml), to which 5% Pd-C (5.0 g)was added, then was stirred vigorously for 115 hours. After filtrationof Pd-C using celite and evaporation of solvent under reduced pressure,the residue was subjected to evaporation under reduced pressure, to givethe object compound (15.66 g) as a colorless oil. Bp. 162°-165° C./0.3mmHg

IR (Neat): 2980, 2940, 1735, 1710, 1607, 848 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.20 (3H,t,J=7 Hz), 1.23 (3H,d, J=6 Hz), 1.30-1.80(4H,m), 1.58 (9H,s), 2.24 (2H,brt,J=6 Hz), 2.77 (1H,dq,J=6 Hz, 6 Hz),4.08 (2H,q,J=7 Hz), 7.20 (2H,d,J=8 Hz), 7.90 (2H,d,J=8 Hz).

Reference Example 18

Production of ethyl 5-[4-(tert-butoxycarbonyl)phenyl]-2-iodohexanate:

The compound (6.41 g) obtained in the Reference Example 17 was subjectedto the same reaction as that in the Reference Example 2 to give theobject compound (3.90 g).

IR (Neat): 2980, 2940, 1738, 1715, 1610, 850 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.23 (3H t J=7 Hz), 1.23 (2H,d,J=7 Hz), 1.40-1.95(4H,m), 1.60 (9H,s), 2.75 (1H,dq,J=6 Hz,6 Hz), 4.15 (2H,q,J=7 Hz), 4.18(1H,t,J=7 Hz), 7.20 (2H,d,J=8 Hz), 7.90 (2H,d,J=8 Hz).

Reference Example 19

Production of ethyl5-[4-(tert-butoxycarbonyl)phenyl]-2-(dicianomethyl)-hexanoate:

The compound (3.90 g) obtained in the Reference Example 18 was subjectedto the same reaction as that in the Reference Example 3 to give theobject compound (3.19 g).

IR (Neat): 2980, 2930, 2250, 1735, 1710, 1605, 847 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.26 (1 5H,t,J=7 Hz), 1.26 (3H,d,J=7 Hz), 1.27(1.5H,t,J=7 Hz), 1.35-1.80 (4H,m), 1.58 (9H,s), 2.50-3.08 (2H,m), 4.00(1H,dd,J=8 Hz,4 Hz), 4.22 (1H,q,J=7 Hz), 4.23 (1H,q,J=7 Hz), 7.18(2H,d,J=8 Hz), 7.92 (2H,d,J=8 Hz).

Working Example 1

Production of tert-butyl4-[3-(2,4-diamino-6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoate:

To a solution of potassium tert-butoxide (2.35 g) and guanidinehydrochloride (1.07 g) in tert-butylalcohol (10 ml) was added a solutionof the compound (3.33 g) obtained in the Reference Example 3 intert-butyl alcohol (30 ml) under an atmosphere of argonr and refluxed byheating for 20 hours. To the reaction mixture were added furtherpotassium tert-butoxide (434 mg) and guanidine hydrochloride (370 mg)and the mixture was refluxed by heating for 8 further hours. Thereaction mixture was cooled, added to 1N potassium hydrogen sulfate inwater (about 10 ml) and adjusted to pH 9. After extraction with atetrahydrofuran-chloroform mixture, the solvent was evaporated off underreduced pressure and the resultant residue was purified by columnchromatography (carrier; silica gel, 100 g, developing solvent;dichloromethane: ethanol=15:1→dichloromethane after mixing withconcentrated aqueous ammonia in a separatory funnel: ethanol=15:1), togive the object compound (1.90 g).

IR (KBr): 3430, 3360, 1710, 1627, 1583, 1432 cm⁻¹.

¹ H-NMR (CDCl₃ -Me₂ SO-d₆) δ: 1.15-1.73 (2H,m), 1.55 (9H,s), 1.73-2.10(2H,m), 2.61 (2H,t,J=7 Hz), 3.35 (1H,t,J=6 Hz), 5.40 (2H,brs), 5.51(2H,brs), 6.30 (1H,brs), 7.12 (2H,d,J=8 Hz), 7.29 (2H,d,J=8 Hz).

Working Example 2

Production of tert-butyl4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoate:

To a solution of the compound (575 mg) obtained in the Working Example 1in tetrahydrofuran (6 ml) was added a solution of borane-tetrahydrofurancomplex (7.5 mmol) in tetrahydrofuran (7.5 ml) at 0° C., and stirred for4.5 hours. To the reaction mixture was added acetic acid-methanol (1:1,6 ml) and the mixture was stirred at room temperature for 15 hours. Thesolvent was evaporated off under reduced pressure and the residue waspurified by column chromatography (carrier; silica gel, 30 g, developingsolvent; dichloromethane: ethanol=100:6 →100:7→100:8→10:1→8:1), to givethe object compound (263 mg).

IR (KBr): 3335, 3180, 2975, 2935, 1710, 1607, 1287, 1163, 1110 cm⁻¹.

¹ H-NMR (Me₂ SO-d₆) δ: 1.54 (9H,s), 1.77-1.90 (2H,m), 2.68(2H,t,J=8 Hz),2.72 (2H,t,J=8 Hz), 5.54 (2H,brs), 6.11 (2H,brs), 6.45 (1H,s), 7.33(2H,d,J=8 Hz), 7.82 (2H,d,J=8 Hz), 10.51 (1H,s).

Working Example 3

Production of diethylN-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamate:

To the compound (381 mg) obtained in the Working Example 2 was addedtrifluoroacetic acid (3 ml) and the mixture was stirred at roomtemperature for 3 hours. Trifluoroacetic acid was evaporated off underreduced pressure, and to the residue obtained by drying at 70° C. underreduced pressure and a solution of diethyl L-glutamate hydrochloride(748 mg) in dimethylformamide (4 ml) was added a solution ofdiphenylphosphoryl azide (858 mg) in dimethylformamide (4 ml) at 0° C.,and then a solution of triethylamine (631 mg) in dimethylformamide (4ml) dropwise at the same temperature. After stirring at 0° C. for 30minutes and then at room temperature for 63 hours, the precipitate wasfiltered off. The solvent was evaporated off under reduced pressure andthe resultant residue was purified by column chromatography (carrier;silica gel, 20 g, developing solvent; dichloromethane after mixing withconcentrated aqueous ammonia in a separatory funnel→dichloromethaneafter mixing with concentrated aqueous ammonia: ethanol=40:1→30:1), togive the object compound (260 mg).

IR (KBr): 3330, 3160, 1735, 1632, 1575, 1540, 1500, 1200 cm⁻¹.

¹ H-NMR (Me₂ SO-d₆) δ: 1.17(3H,t,J=7 Hz), 1.20 (3H,t,J=7 Hz), 1.80-2.20(4H,m), 2.44 (2H,t,J=7 Hz), 2.68 (2H,t, J=7 Hz), 2.72 (2H,t,J=7 Hz),4.05 (2H,q,J=7 Hz), 4.11 (2H,q,J=7 Hz), 4.35-4.80 (1H,m), 5.34 (2H,s),5.91 (2H,s), 6.42 (1H,s), 7.31 (2H,d,J=8 Hz), 7.80 (2H,d,J=8 Hz), 8.86(1H,d,J=8 Hz), 10.51 (1H,s).

Working Example 4

Production of N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid:

The compound (250 mg) obtained in the Working Example 3 was dissolved intetrahvdrofuran-water mixture (1:1, 7 ml), to which 1N sodium hydroxidein water (2.52 ml) was added and the mixture was stirred at roomtemperature for 1.5 hours. The solution was concentrated to 3 ml underreduced pressure, and the resultant insoluble matter was filtrated offthrough a millipore filter. To the filtrate, cooled to 0° C., was addedacetic acid (0.5 ml) and the resultant crystals were collected byfiltration and washed throughly with ice-water. The crystals weredried-at 70° C. under reduced pressure, to give the object compound (201mg) as white crystals.

IR (KBr): 3340, 3200, 2940, 1660-1630, 1540, 1500, 1397 cm⁻¹.

¹ H-NMR (Me₂ :SO-d₆) δ: 1.75-2.20 (4H,m), 2.35 (2H,t,J=7 Hz), 2.68(2H,t,J=7 Hz), 2.71 (2H,t,J=7 Hz), 4.30-4.47 (1H,m), 5.53 (2H,brs), 6.15(2H,s), 6.46 (1H,s), 7.31 (2H,d,J=8 Hz), 7.81 (2H,d,J=8 Hz), 8.48(1H,d,J=8 Hz); 10.51 (1H,s).

Working Example 5

Production of diethylN-[4-[3-(2,4-diamino6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamate:

The compound (200 mg) obtained in the Working Example 1 was subjected tothe same reaction as that in the Working Example 3, to give the objectcompound (164 mg).

IR (KBr): 3355, 3230, 2995, 2990, 1740, 1638, 1595, 1590 cm⁻¹.

¹ H-NMR (CDCl₃ /Me₂ SO-d₆) δ: 1.20 (3H,t,J=7 Hz), 1.25 (3H,t, J=7 Hz),1.25-2.70 (11H,m), 3.25-3.45 (1H,m), 4.0 5 (2H,q,J=7 Hz), 4.15 (2H,q,J=7Hz), 4.38-4.68 (1H,m), 5.63 (2H,brs), 5.66 (2H, brs), 7.16 (2H,d, J=8Hz), 7.76 (2H, d, J=8 Hz), 8.39 (1H,d, J=8 Hz), 10.50 (1H,s).

Working Example 6

Production ofN-[4-[3-(2,4-diamino-6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glumaticacid:

The compound (112 mg) obtained in the Working Example 5 was subjected tothe same reaction as that in the Working Example 4, to give the objectcompound (60 mg).

IR (KBr): 3350, 3210, 2950, 1730, 1660, 1630 cm⁻¹.

¹ H-NMR (Me₂ SO-d₆) δ: 1.20-1.56 (2H,m), 1.65-2.20(4H,m), 2.35 (2H,t,J=7Hz), 2.50-2.65 (2H,m), 3.25-3.35 (1H,m), 4.32-4.46 (1H,m), 5.90(2H,brs), 6.00 (2H,brs). 7.22 (2H,d,J=8 Hz), 7.78 (2H,d,J=8 Hz), 8.52(1H,d,J=8 Hz), (1H,s).

Working Example 7

Production of diethytN-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamate:

The compound (150 mg) obtained in the Reference Example 7 was dissolvedin ethanol (22.5 ml), to which were added 10% Pd-C (450 mg: manufacturedby Engelhand Co. Ltd.) and 2 drops of acetic acid and stirred vigorouslyat room temperature for 62.5 hours. The catalyst was filtered off, thefiltrate was concentrated to dryness and the residue was purified bycolumn chromatography (carrier; silica gel, 10 g, developing solvent;chloroform containing 5% ethanol), to give the object compound (33 mg).

IR (KBr): 3340, 2940, 1740, 1680, 1630, 1540, 1440, 1380, 1340, 1210,1100, 1020, 860 cm⁻¹.

¹ H-NMR (CDCl₃ /CD₃ OD) δ: 1.20 (3H,t,J=6 Hz), 1.28 (3H,t,J=6 Hz),1.87-2.36 (4H,m), 2.40-2.57 (2H,m), 2.60-2.87 (4H,m), 3.96-4.37 (4H,q x2,J=6 Hz), 4.56-4.90 (1H,m), 6.37 (1H,s), 7.23 (2H,d,J=7.5 Hz), 7.71(2H,d,J=7.5 Hz).

Working Example 8

Production ofN-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamicacid:

The compound (27 mg) obtained in the Working Example 7 was dissolved ina tetrahydrofuran-water mixture (1:1, 2.16 ml), to which was added 1Nsodium hydroxide in water (0.189 ml) and stirred at room temperature for2.5 hours. A large portion of tetrahydrofuran was evaporated off, aceticacid (0.189 ml) was added by ice-cooling and stirred. The resultingprecipitate was collected by filtration and dried, to give the objectcompound 19 mg)

IR (KBr): 3400, 3300, 2950, 1700, 1650, 1540, 1510, 1400, 1340, 1240,1080, 1020 cm⁻¹

¹ H-NMR (Me₂ SO-d₆) δ: 1.80-2.17 (4H,m), 2.23-2.40 (2H,m), 2.53-2.83(4H,m), 4.27-4.56 (1H,m), 6.33 (1H,s), 7.27 (2H,d,J=7.5 Hz), 7.78(2H,d,J=7.5 Hz).

Working Example 9

Production of tert-butyl4-[3-(2,4-diamino-6-oxo-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoate:

To a solution of potassium tert-butoxide (2.35 g) and guanidinehyrochloride (1.07 g) in tert-butyl alcohol (10 ml) was added a solutionof the compound (3.33 g) obtained in the Reference Example 16 intert-butyl alcchol (30 ml) under an atmosphere of argon, and refluxed byheating for 20 hours. To the reaction mixture were added furtherpotassium tert-butoxide (434 mg) and guanidine hydrochloride (370 mg),and refluxed by heating for further 8 hours. The reaction mixture wascooled, added to 1N potassium hydrogen sulfate in water (about 10 ml),to be adjusted to pH 9. After extraction with atetrahydrofuran-chloroform mixture, the solvent was evaporated off underreduced pressure, and the resultant residue was purified by columnchromatography (carrier; silica gel, 100 g, dichloromethane-ethanol,15:1 dichloromethane after mixing with concentrated aqeous ammonia in aseparatory funnel-ethanol, 15:1), to give the object compound (1.90 g).

IR (KBr): 3430, 3360, 1710, 1527, 1583, 1432 cm⁻¹.

¹ H-NMR (CDCl₃ /Me₂ SO-d₆) δ: 1.15-1.73 (2H,m), 1.55 (9H,s), 1.73-2.10(2H,m), 2.61 (2H,t,J=7 Hz), 3.35 (1H,t,J=6 Hz), 5.40 (2H,brs), 5.51(2H,brs), 6.30 (1H,brs), 7.12 (2H,d,J=8 Hz), 7.29(2H,d,J=8 Hz).

Working Example 10

Production of tert-butyl4-[3-(2,4-diamino-5,6-dihydropyrroio[2,3-d]pyrimidin-5-yl)propyl]benzoate:

To a solution of the compound (430 mg) obtained in the Working Example 9in tetrahydrofuran (10 ml) was added a solution ofborane-tetrahydrofuran complex (16.8 mmol) in tetrahydrofuran (10 ml)and the mixture was refluxed by heating for 4 hours. After cooling, thereaction mixture was added to ice water and stirred vigorously at pH 2(adjusted by addition of 1N hydrochloric acid) for 3 minutes and then atpH 10.5 (adjusted by addition of 2N potassium carbonate in water) for 5minutes. The reaction mixture was extracted withtetrahydrofuran-chloroform mixture. The solvent was evaporated off underreduced pressure, and the residue was purified by column chromatography(carrier; silica gel, 20 g, dichloromethane-ethanl, 30:1→15:1dichloromethane after mixing with concentrated aqueous ammonia in aseparatory funnel-ethanol, 20:1), to give the object compound (114 mg).

IR (KBr): 3375, 3325, 3190, 2970, 2930, 1712, 1603 cm⁻¹.

¹ H-NMR (CDCl₃ /Me₂ SO-d₆) δ: 1.45-2.15 (4H,m), 1.57 (9H,s), 2.65(2H,t,J=7 Hz), 3.00-3.28 (2H,m), 3.44-3.70 (1H,m), 4.85 (2H,brs), 4.90(2H,brs), 5.30 (1H,brs), 7.19 (2H,d,J=8 Hz), 7.80 (2H,d,J=8 Hz).

Working Example 11

Production of diethylN-[4-[3-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamate:

The compound (1.47 g) obtained in the Reference Example 10 was suspendedin tetrahydrofuran (60 ml), to which 0.1N sodium hydroxide in water (120mt) was added, and the mixture was stirred at room temperature for 2hours. Then the solution was neutralized with 0.1N hydrochloric acid (60ml) and concentrated to dryness under reduced pressure. The residue wassuspended in dried dimethylformamide (112.5 ml), to which diethylL-glutamate hydrochloride (2.88 g), diphenylphosphoryl azide (1.295 ml)and triethylamine (2.52 ml) were added. The mixture was brought back tothe room temperature, and stirring was continued for 63 hours. Theresulting precipitate was filtered off, and the filtrate wasconcentrated to dryness under reduced pressure. The residue wassubjected to separation-purification with column chromatography onsilica gel (carrier; 100 g) (ethanol containing 6.9% ammonia-chloroformcontaining 6.9% ammonia, 1:20→1:10), to give the object compound (1.12g) as a colorless powder.

IR (KBr): 3330, 2930, 1740, 1670, 1640, 1570, 1540, 1440, 1375, 1300,1200, 1095, 1020 cm⁻¹.

¹ H-NMR (CDCl₃ /CD₃ OD) δ: 1.20 (3H,t,J=7.5 Hz), 1.27 (3H,t,J=7.5 Hz),1.47-1.83 (4H,m), 2.00-2.36 (2H,m), 2.37-2.50 (2H,m), 2.67 (2H t J=7.5Hz), 3.10-3.37 (2H m), 3.53-3.80 (1H,m), 3.96-4.33 (4H,qx2,J=7.5 Hz),4.60-4.87 (1H,m), 7.25 (2H,d,J=9 Hz), 7.75 (2H,d,J=9 Hz).

Working Example 12

Prcduction of diethylN-[4-[2-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]L-glutamate:

The compound (0.315 g) obtained in the Reference Example 13 wassubjected to the same reaction as that in the Working Example 11, togive the object compound (0.247 g).

IR (KBr): 3310, 2990, 1740, 1730, 1690, 1640, 1570, 1530, 1440, 1375,1330, 1300, 1240, 1200, 1090, 1010, 850 cm⁻¹.

¹ H-NMR (CDCl₃ /CD₃ OD) δ: 1.22 (3H,t,J=7.5 Hz), 1.30 (3H,t,J=7.5 Hz),1.53-2.87 (8H,m), 3.13-3.90 (3H,m), 4.00-4.43 (4H,qx2,J=7.5 Hz),4.57-4.90 (1H,m), 7.25 (2H,d,J=9 Hz), 7.72 (2H,d,J=9 Hz).

Working Example 13

Production of diethylN-[4-[3-(2,4-diamino-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]L-glutamate:

The compound (94 mg) obtained in the Working Example 10 was dissolved in1 ml of trifluoroacetic acid and stirred at room temperature for 3hours. The solvent was evaporated off under reduced pressure, and to themixture of the residue obtained by drying at 70° C. under reducedpressure and diethyl L-glutamate (304 mg) in dimethylformamide (2 ml),was added a solution of diphenylphosphoryl azide (350 mg) indimethylformamide (1.5 ml) at 0° C., and then a solution oftriethylamine (180 mg) in dimethylformamide (1.5 ml) dropwise at thesame temperature. After stirring at 0° C. for 30 minutes and then atroom temperature for 78 hours, the solvent was evaporated off underreduced pressure. The resultant residue was purified by columnchromatography (carrier; silica gel, 20 g, dichloromethane after mixingwith concentrated aqueous ammonia in a separatory funnel→dichloromethaneafter mixing with concentrated aqueous ammonia --ethanol 40:1→30:1), togive the object compound (88 mg).

IR (KBr): 3350, 2990, 2945, 1740, 1610, 1540, 1508, 1438 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.23 (H,g,J=7 Hz), 1.43-1.80 (3H,m), 1.85-2.77(7H,m), 2.95-3.30 (2H,m), 3.58 (1H,t,J=11 Hz), 4.07 (2H,q,J=7 Hz), 4.20(2H,q,J=7 Hz), 4.25 (1H,brs), 4.63-4.83 (1H,m), 4.68 (1H,brs), 7.00-7.23(1H,m), 7.13 (2H,d,J=8 Hz), 7.67 (2H,d,J=8 Hz).

Working Example 14

Production ofN-[4-[3-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]henzoyl]L-glutamicacid:

The compound (1.05 g) obtained in the Working Example 11 was dissolvedin tetrahydrofuran-water mixture (2:1, 63 ml), to which 1N sodiumhydroxide in water (7.35 ml) was added, and stirred at room temperaturefor 2.5 hours. Tetrahydrofuran was evaporated off, a small amount ofinsoluble matter was filtrated off, acetic acid (7.35 ml) was added tothe filtrate, and the resulting precipitate was collected by filtration.The precipitate was washed. with water and dried, to give the objectcompound (0.85 g) as a colorless powder.

IR (KBr): 3340, 2930, 1690, 1630, 1540, 1440, 1300, 1080, 850 cm⁻¹.

¹ H-NMR (DMSO-d₆) δ: 1.20-1.80 (4H,m), 1.87-2.17 (2H,m), 2.23-2.40(2H,m), 2.50-2.77 (2H,m), 2.83-3.20 (2H,m), 3.30-3.63 (1H,m), 4.23-4.53(1H,m), 7.26 (2H,d,J=9 Hz), 7.77 (2H,d,J=9 Hz).

Working Example 15

Production ofN-[4-[2-(2-amino-4-hydroxy-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamicacid:

The compound (0.195 g) obtained in the Working Example 12 was subjectedto the same reaction as that in the Working Example 14, to give theobject compound (0.153 g).

IR (KBr): 3250, 2900, 1650, 1580, 1440, 1300, 1090 cm⁻¹.

¹ H-NMR (DMSO-d₆) δ: 1.43-1.76 (1H,m), 1.98 (2H,t,J=7.5 Hz), 1.80-2.10(1H,m), 2.13-2.40 (2H,m), 2.67 (2H,t,J=9 Hz), 2.90-3.23 (2H,m),3.33-3.60 (1H,m), 4.10-4.43 (1H,m), 7.28 (2H,d,J=9 Hz), 7.75 (2H,d,J=9Hz).

Working Example 16

Production ofN-[4-[3-(2,4-diamino-5,6-dihydropyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]L-glutamticacid

The compound (41 mg) obtained in the Working Example 13 was subjected tothe same reaction as that in the Working Example 14, to give the objectcompound (32 mg).

IR (KBr): 3700-2350, 3215, 1690-1620, 1540 cm⁻¹.

¹ H-NMR (Me₂ SO -d₆) δ: 1.02-1.85 (4H,m), 1.85-2.83 (6H,m), 2.90-3.30(2H,m), 3.55 (1H,t,J=11 Hz), 4.15-4.45 (1H,m), 6.38 (2H,brs), 6.77(2H,brs), 6.90 (1H,brs), 7.22 (2H,d,J=8 Hz), 7.74 (2H,d,J=8 Hz), 8.22(1H,d,J=7 Hz).

Working Example 17

Production of tert-butyl4-[3-(2,4-diamino-6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoate:

The compound (3.18 g) obtained in the Reference Example 19 was subjectedto the same reaction as that in the Working Example 1, to give theobject compound (2.61 g).

IR (KBr): 3360, 3235, 2975, 2700, 1715, 1625, 1584 1438, 1290, 1163,1118, 848 cm⁻¹.

¹ H-NMR (Me₂ SO-d₆) δ: 1.14 (3H,d,J=7 Hz), 1.20-1.50 (2H,m), 1.54(9H,s), 1.55-1.80 (1H,m), 1.80-2.05 (1H,m), 2.60-2.78 (1H,m), 3.20-3.30(1H,m), 5.86 (2H,brs), 5.96 (2H,brs), 7.25 (2H,d,J=8 Hz), 7.81 (2H,d,J=8Hz), 10.42 (1H,s).

Working Example 18

Production of tert-butyl4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoate(A) andtert-butyl-4-[3-(2,4-diamino-7H-pyrrolo-[2,3-d]pyrimidin-5-yl)-1-methyl-propyl]benzoate(B):

To a suspension of the compound (2.00 g) obtained in the Working Example17 in tetrahydrofuran (25 ml) was a solution of boiane-tetrahydrofurancomplex (40.3 mmol) tetrahydrofuran (40.3 ml). After stirring for 10minutes, the mixture was cooled to room temperature and further stirredfor 5 hours. To the reaction mixture was added an acetic acid-methanolmixture (1:1, 40 ml) and the mixture was stirred for 18 hours at roomtemperature. After evaporation of solvent under reduced pressure, theresulting residue was purified by column chromatography (carrier: silicagel, 100 g, developing solvent; dichloromethane-ethanol=20:1→25:2, thendichloromethane-ethanol containing ammonia (6%)), to give the objectcompound (A) (579 mg) and (B) (1.214 g).

(A) IR (KBr): 3350, 3200, 2980, 2940m, 1714, 1650, 1608, 1290, 1163,1115, 848 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.31 (3H,d,J=7 Hz), 1.60 (9H,s), 1.94 (2H,dt,J=8 Hz,8Hz), 2.40-2.60 (2H,m), 2.85 (1H,tq,J=7 Hz,7 Hz) , 4.50-5.50 (4H,br),6.46 (1H,s), 7.27 (2H,d,J=8 Hz), 7.96 (2H,d,J=8 Hz), 9.20 (1H,brs).

(B) IR (KBr): 3340, 3195, 2980, 2935, 1715, 1507, 1430, 1295, 1163,1115, 847 cm⁻¹.

¹ H-NMR (Me₂ SO-d₆) δ: 1.18 (2.25H,d,J=7 Hz), 1.19 (0.75H,d,J=7 Hz),1.23-1.42 (2H,m), 1.45-1.65 (2H,m), 1.54 (9H,s), 2.54-2.70 (1H,m),2.90-3.08 (2H,m), 3.30-3.50 (1H,m), 5.43 (4H,s), 5.95 (0.25H,s), 6.00(0.75H,s), 7.32 (2H,d,J=8 Hz), 7.82 (2H,d,J=8 Hz).

Working Example 19

Production of diethylN-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]-L-glutamate

The compound (A) (581 mg) obtained in the Working Example 18 wassubjected to the same reaction as that in the Working Example 3 to givethe object compound (640 mg).

IR (KBr): 3375, 3200, 2975, 2930, 1738, 1608, 1430 1200, 1008, 853 cm⁻¹.

¹ H-NMR (Me₂ SO-d₆) δ: 1.17 (3H,t,J=7 Hz), 1.18 (3H,d,J=7 Hz), 1.19(3H,t,J=7 Hz), 1.26-1.44 (2H,m), 1.44-1.63 (2H,m), 1.90-2.20 (2H,m),2.44 (2H,t,J=7 Hz), 2.63-2.80 (1H,m), 2.90-3.08 (2H,m), 3.30-3.50(1H,m), 4.05 (2H,q,J=7 Hz), 4.11 (2H,g,J=7 Hz), 4.37-4.50 (1H,m), 5.36(2H, s), 5.37 (2H,s), 5.87 (0.25H,s), 5.91 (0.75H,s), 7.30 (2H,d,J=8Hz), 7.80 (2H,d,J=8 Hz), 8.66 (1H,d,J=8 Hz).

Working Example 20

Production ofN-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)-t-methylpropyl]benzoyl]-L-glutamicacid:

The compound (600 mg) obtained in the working Example 19 was subjectedto the same reaction as that in the Working Example 4, to give theobject compound (508 mg).

IR (KBr): 3350, 3200, 1690, 1680-1610, 1635, 1530, 1400, 1300, 853 cm⁻¹.

¹ H-NMR (Me=SO-d₆) δ: 1.20 (3H,d,J=7 Hz), 1.25-1.65 (4H,m), 1.87-2.20(2H,m), 2.30 (2H,t,J=7 Hz), 2.60-2.80 (1H,m), 3.00-3.20 (2H.,m),3.42-3.60 (1H,m), 4.22-4.40 (1H 6.20-5.08 (5H,m), 7.28 (2H,d,J=8 Hz),7.78 (2H,d,J=8 Hz), 8.28-8.36 (1H,m).

Working Example 21

Production of diethylN-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]L-glutamicacid:

The compound (B) (540 mg) obtained in the Working Example 18 wassubjected to the same reaction as that in the Working Example 3, to givethe object compound (556 mg).

IR (KBr): 3340, 3180, 2935, 1735, 1640, 1610, 1580, 1200, 1095, 1018,850 cm⁻¹.

¹ H-NMR (CDCl₃) δ: 1.23 (3H,t,J=7 Hz), 1.30 (3H,d,J=7 Hz), 1.31(3H,t,J=7 Hz), 1.80-2.05 (4H,m), 2.15-2.57 (4H,m), 2.83 (1H,tq,J=7 Hz,7Hz), 4.12 (2H,q,J=7 Hz), 4.25 (2H,q,J=7 Hz), 4.68 (2H,brs), 4.75-4.87(1H,m), 4.92 (2H,brs), 6.43 (1H,s), 7.26 (2H,d,J=8 Hz), 7.37 (1H,dd,J=7Hz,3 Hz), 7.77 (2H,d,J=8 Hz), 8.81 (1H,brs).

Working Example 22

Production ofN-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-t-methylpropyl]benzoyl]-L-glutamicacid:

The compound (533 mg) obtained in the Working Example 21 was subjectedto the same reaction as that in the Working Example 4, to give theobject compound (436 mg).

IR (KBr): 3350, 3205, 1650, 1540, 1540, 1400, 850 cm⁻¹.

¹ H-NMR (Me₂ SO-d₆) δ: 1.25 (3H,d,J=7 Hz), 1.73-2.20 (4H,m), 2.35(3H,t,J=8 Hz), 2.40-2.68 (2H,m), 2.85 (1H,tg,J=7 Hz,7 Hz), 4.32-4.45(1H,m), 5.54 (2H,brs), 6.06 (2H,brs), 6.38 (1H,s), 7.33 (2H,d,J=8 Hz),7.83 (2H,d,J=8 Hz), 8.49 (1H,d,J=8 Hz), 10.45 (1H,s).

Working Example 23

The compound (50 mg per tablet) obtained in the Working Example 14,lactose (250 mg per tablet), corn starch (51 mg per tablet) andhydroxypropylcellulose L (9 mg per tablet) were mixed according to theconventional method and granulated. The granules, corn starch (8 mg pertablet) and magnesium stearate (2 mg per tablet) were mixed andtabletted according to the conventional method, to give tablets (370 mgper tablet).

Working Example 24

Ten grams of the sodium salt of the compound obtained in the WorkingExample 14 was dissolved in 1 l of physiological saline. The solutionwas filerted through a microfilter and dispensed in 2.2 ml aliquots inampoules, sterilized at 110° C. for 30 minutes and the ampoules may beused for subcutaneous, intravenous or intramuscular injections.

Working Example 25

Five grams of the hydrochloride of the compound obtained in the WorkingExample 14 and 10 g of mannitol were dissolved in 1 l of distilledwater, and the solution was dispensed in 2 ml aliquots into ampulesafter filtration through a bacterial filter. The ampoules were dried ina freeze-drier and sealed, and thus the ampoules of which content isdissolved before use were obtained. Before use for injection, theampoules are opened and the content is dissolved in, for example, 2 mlof physiological saline.

What is claimed is:
 1. A method for producing a compound of the formula##STR12## wherein the ring A is a pyrrole or pyrroline ring, X is anamino group or a hydroxyl group, Y is a hydrogen atom, an amino group ora hydroxyl group, R is a hydrogen atom, a fluorine atom, an alkyl group,an alkenyl group or an alkynyl group, --COOR¹ and COOR² are a carboxylgroup which may be esterified and n is an integer of 2 to 4, and R maybe different in each of the n repeating units, or salts thereof, whichcomprises reacting a compound of the formula ##STR13## wherein the ringA, X, Y, R and n are the same as defined above, a reactive derivative atthe carboxyl group or a salt thereof, with a compound of the formula##STR14## wherein --COOR¹ and --COOR² are the same as defined above.